We are developing HPIVs and APMVs as vaccine vectors for human use against highly pathogenic emerging viruses, using HPIV3 and NDV as proof of principle. Regarding the HPIVs, we previously showed that a single immunization of African green monkeys with a PIV3-based vector (an attenuated chimera of HPIV3 and BPIV3 called B/HPIV3) expressing the SARS spike S glycoprotein induced immunity sufficient to prevent shedding by a high-dose IN/IT challenge of SARS-CoV. We also previously showed that a single immunization of rhesus monkeys with HPIV3 expressing the single glycoprotein GP of Ebola virus (EV) was 78% effective in preventing mortality from an EV challenge, and two doses provided sterilizing immunity and protection in all of the animals. We presently are evaluating the aerosol route for administration of HPIV3/GP vaccine in rhesus monkeys, and also are comparing this vaccine versus an alphavirus vector expressing GP. Most older children and adults have immunity against the HPIVs due to natural exposure, which could restrict infectivity and immunogenicity, although it is characteristic of the HPIVs that protection is incomplete. However, we showed that a single immunization of rhesus monkeys that previously had been infected twice with HPIV3 resulted in titers of EV-specific serum antibodies that were substantially less than in HPIV3-nave animals, but the titers induced by a second dose were equivalent to those induced by two doses in HPIV-nave animals. Thus, the use of HPIV vectors in HPIV-experienced individuals may be feasible. We also created a version of HPIV3/GP in which the HPIV3 F and HN genes were deleted, leaving EV GP as the sole viral surface antigen. In guinea pigs, this virus was very highly attenuated, but a single immunization induced sterilizing immunity against EV challenge. This virus presently is being evaluated in rhesus monkeys. A second strategy has been to investigate APMVs as vectors, in particular NDV. We previously showed that NDV is naturally restricted in non-human primates due to host range differences. Indeed, most immunized animals did not detectably shed NDV, and direct analysis of lung tissue revealed very low levels of replication. NDV-based vectors expressing protective antigens of Ebola virus, SARS, or HPAIV were immunogenic and protective against the respective pathogen in non-human primates, although two doses were needed due to the high level of restriction. We have investigated ways to increase NDV replication to obviate the need for two doses. We previously described improved replication based on modifying the F protein cleavage site or removing N-glycans from the heptad repeats of the F protein. In the present report, we investigated the effect of removing or mutating residues in the cytoplasmic tail (CT) of the F protein. The CT of the NDV F protein is 31 residues long (amino acid 523 to 553), and studies with transfected plasmids have indicated a role for the CT in fusion. We used reverse genetics to investigate the effects of mutations in the F protein CT in the context of complete infectious virus, using the moderately pathogenic NDV strain Beaudette C. Specifically, we monitored effects on F protein cell surface expression, fusion, viral replication in vitro and in vivo, and pathogenesis in a natural host. Out of a series of progressively longer C-terminal deletions in the CT, we were able to rescue recombinant viruses lacking two or four residues (r&#8710;2 and r&#8710;4). We also generated and rescued mutants with individual amino acid substitutions at each of these four terminal residues (rM553A, rK552A, rT551A, rT550A). In addition, the NDV F CT has two conserved tyrosines (Y524 and Y527) and a di-leucine motif (LL536-537). In other paramyxoviruses, these residues were shown to affect fusion activity and are a central element in basolateral targeting signals thus modulating viral pathogenesis. We successfully rescued recombinant viruses with substitution of either tyrosine residue (rY524A and rY527A), but could not recover virus with substitution mutations in the di-leucine motif. With the exception of one mutant (rT550A) that closely resembled wild-type virus (rWT), these mutant viruses exhibited increased cell surface expression of the F protein, were hyperfusogenic, and had increased replication in vitro and in vivo and increased pathogenicity in embryonated chicken eggs, 1-day-old chicks, and 2-week-old chickens. The fusion efficiency showed moderate dependency on the expression level of mutant F protein. We conclude that these residues in the F CT have the effect of down-regulating fusion and virulence. These mutations may assist in the development of NDV as a vaccine vector. We also are evaluating other serotypes of APMV as potential vectors. We previously determined the complete genome sequences for serotypes 2-9 (NDV is serotype 1). We have evaluated the role of the F protein cleavage site in the replication and pathogenicity of APMV4. This was done based on the paradigm of NDV, for which a multi-basic cleavage site in F is associated with increased virulence whereas a single basic residue is associated with avirulence. We constructed a reverse genetics system for recovery of infectious recombinant APMV-4 from cloned cDNA. The recovered recombinant APMV-4 resembled the biological virus in growth characteristics in vitro and in low pathogenicity in eggs and chickens. The F cleavage site sequence of APMV-4 (DIQPR&#8595;F) contains a single basic amino acid, at the -1 position. Six mutant APMV-4 viruses were designed and recovered in which the F protein cleavage site was mutated to contain increased numbers of basic amino acids or to mimic the naturally occurring cleavage sites of several paramyxoviruses, including neurovirulent and avirulent strains of NDV. The presence of a glutamine residue at the -3 position was found to be important for mutant virus recovery. In addition, cleavage sites containing the furin protease motif conferred increased replication and syncytium formation in vitro. However, analysis of viral pathogenicity in 9-day-old embryonated chicken eggs, 1-day-old and 2-week-old chickens, and 3-week-old ducks showed that none the F protein cleavage site mutations altered the replication, tropism, and pathogenicity of APMV-4, and no significant differences were observed among the parental and mutant APMV-4 viruses in vivo. Although parental and mutant viruses replicated somewhat better in ducks than in chickens, they all were highly restricted and avirulent in both species. These results suggested that the cleavage site sequence of the F protein is not a limiting determinant of APMV-4 pathogenicity in chickens and ducks. Therefore, a feature that is a major determinant of virulence for NDV does not have the same significance for APMV4. This is of general interest, since the paramyxovirus contains a wide array of pathogens of humans and animals (e.g., the HPIVs, RSV, HMPV, mumps, measles, rinderpest, Nipah, Hendra viruses), and information on the mechanisms of pathogenesis for any of these viruses is important.